Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long-Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

GPCRs such as 5-HT2A and D2 are implicated in the therapeutic and the side effects of antipsychotics. However, the pattern of brain activity that leads to the behavioral effects of antipsychotics is poorly understood. To address this question, we used the transgenic 'FosTRAP' mice (Mus musculus), where a fluorescent reporter marks the cells responsive to the stimulus of interest. Here, the stimulus was an administration of various antipsychotic drugs. In case of typical antipsychotics such as Haloperidol, the c-fos active cells were predominantly found in the striatum, whereas in case of the atypical antipsychotics (Clozapine and Olanzapine), c-fos-induced cells were more numerous in the cortical regions, e.g., orbital cortex, piriform cortex. Curiously, we also observed ependymal cells to be a novel cellular target of atypical antipsychotics. 5-HT2A is considered to be a major target for atypical antipsychotics. Therefore, we bred 'FosTRAP' mice with 5-HT2A knock-out (KO) mice and tested their response to the prototype of atypical antipsychotics, Clozapine. Interestingly, the absence of 5-HT2A did not significantly affect the number of c-fos-induced cells in the cortical regions. However, the ependymal cells showed a dramatically reduced response to Clozapine in the absence of 5-HT2A. In summary, the TRAP system has allowed us to identify various region-specific activity induced by antipsychotics and novel cellular targets of the antipsychotics. These results serve as a "proof of principle" study that can be extended to explore the biochemical and physiological changes brought about by antipsychotics and specifically identify antipsychotic-responsive cells in the live tissue.

Manipulation of neuronal activity during the early postnatal period in monkeys has been largely limited to permanent lesion studies, which can be impacted by developmental plasticity leading to reorganization and compensation from other brain structures that can interfere with the interpretations of results. Chemogenetic tools, such as DREADDs (designer receptors exclusively activated by designer drugs), can transiently and reversibly activate or inactivate brain structures, avoiding the pitfalls of permanent lesions to better address important developmental neuroscience questions. We demonstrate that inhibitory DREADDs in the amygdala can be used to manipulate socioemotional behavior in infant monkeys. Two infant rhesus monkeys (1 male, 1 female) received AAV5-hSyn-HA-hM4Di-IRES-mCitrine injections bilaterally in the amygdala at 9 months of age. DREADD activation after systemic administration of either clozapine-N-oxide or low-dose clozapine resulted in decreased freezing and anxiety on the human intruder paradigm and changed the looking patterns on a socioemotional attention eye-tracking task, compared with vehicle administration. The DREADD-induced behaviors were reminiscent of, but not identical to, those seen after permanent amygdala lesions in infant monkeys, such that neonatal lesions produce a more extensive array of behavioral changes in response to the human intruder task that were not seen with DREADD-evoked inhibition of this region. Our results may help support the notion that the more extensive behavior changes seen after early lesions are manifested from brain reorganization that occur after permanent damage. The current study provides a proof of principle that DREADDs can be used in young infant monkeys to transiently and reversibly manipulate behavior.

Stress-inducing events during pregnancy are associated with aberrant neurodevelopment resulting in adverse psychiatric outcomes, including autism spectrum disorder (ASD). While numerous preclinical models for the study of ASD are frequently generated using C57BL/6J mice, few studies have investigated the effects of prenatal stress on this genetic background. In the current manuscript, we stressed C57BL/6 dams during gestation and examined numerous behavioral and molecular endophenotypes in the adult male and female offspring to characterize the resultant phenotype as compared with offspring born from nonstressed (NS) dams. Adult mice born from prenatal restraint stressed (PRS) dams demonstrated reduced sociability and reciprocal social interaction along with increased marble burying behaviors relative to mice born from nonstressed control dams. Differential expression of genes related to excitatory and inhibitory neurotransmission was evaluated in the medial prefrontal cortex, amygdala, hippocampus, nucleus accumbens and caudate putamen via qRT-PCR. The male PRS mouse behavioral phenotype coincided with aberrant expression of glutamate and GABA marker genes (e.g., Grin1, Grin2b, Gls, Gat1, Reln) in neural substrates of social behavior. Rescue of the male PRS sociability deficit by a known antipsychotic with epigenetic properties (i.e., clozapine (5 mg/kg) + 18 hr washout) indicated possible epigenetic regulation of genes that govern sociability. Clozapine treatment increased the expression levels of genes involved in DNA methylation, histone methylation, and histone acetylation in the nucleus accumbens. Identification of etiology-specific mechanisms underlying clinically relevant behavioral phenotypes may ultimately provide novel therapeutic interventions for the treatment of psychiatric disorders including ASD.

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG)/dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced designer receptors exclusively activated by designer drugs (DREADD) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

Goal-tracking (GT) rats are sensitive to Pavlovian outcome devaluation while sign-tracking (ST) rats are devaluation insensitive. During outcome devaluation, GT rats flexibly modify responding to cues based on the current value of the associated outcome. However, ST rats rigidly respond to cues regardless of the current outcome value. Prior work demonstrated disconnection of the basolateral amygdala (BLA) and anterior insular cortex (aIC) decreased both GT and ST behaviors. Given the role of these regions in appetitive motivation and behavioral flexibility, we predicted that disrupting BLA to aIC pathway during outcome devaluation would reduce flexibility in GT rats and reduce rigid appetitive motivation in ST rats. We inhibited the BLA to aIC pathway by infusing inhibitory DREADDs (hM4Di-mcherry) or control (mCherry) virus into the BLA and implanted cannulae into the aIC to inhibit BLA terminals using intracranial injections of clozapine N-oxide (CNO). After training, we used a within-subject satiety-induced outcome devaluation procedure in which we sated rats on training pellets (devalued condition) or homecage chow (valued condition). All rats received bilateral CNO infusions into the aIC before brief nonreinforced test sessions. Contrary to our hypothesis, BLA-IC inhibition did not interfere with devaluation sensitivity in GT rats but did make ST behaviors sensitive to devaluation. Intermediate rats showed the opposite effect, showing rigid responding to cues with BLA-aIC pathway inactivation. Together, these results demonstrate BLA-IC projections mediate tracking-specific Pavlovian devaluation sensitivity and highlights the importance of considering individual differences in Pavlovian approach when evaluating circuitry contributions to behavioral flexibility.

Selective neuromodulation using designer receptors exclusively activated by designer drugs (DREADDs) has become an increasingly important research tool, as well as an emerging therapeutic approach. However, the safety profile of DREADD expression is unknown. Here, different titers of adeno-associated viral (AAV) vector were administered in an attempt to vary total expression levels of the inhibitory DREADD hM4D(Gi) in excitatory hippocampal neurons. Male Sprague Dawley rats were injected with AAV2/7 encoding DREADD-mCherry, DREADD, or mCherry. Pronounced neuronal loss and neuroinflammatory reactions were observed after transduction with the high titer DREADD AAV, which also resulted in the highest DREADD expression levels. No such effects were observed in the mCherry control group, despite an equally high titer, nor in conditions where lower viral vector titers were injected. In the high titer DREADD conditions, dentate gyrus (DG) evoked potentials were inhibited on clozapine-induced activation of hM4D(Gi), while in low titer conditions DG evoked potentials were enhanced. Recordings of single neuronal activity nevertheless indicated a reduction in spontaneous firing of granule cell layer neurons. Our results indicate that prolonged, high levels of DREADD expression can have neurotoxic effects and that chemogenetic suppression of excitatory hippocampal neurons can paradoxically enhance DG evoked potentials.

Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

Circadian rhythms are 24-h cycles in physiology regulated by the suprachiasmatic nucleus (SCN) in the brain, where daily cues act on SCN neurons to alter clock timing. Cannabinoid signaling modulates SCN neuronal activity, although the mechanism remains unclear. We propose that neuronal activity generates endocannabinoid release, activating astrocyte Ca2+ signaling, which releases adenosine and activates adenosine-1 receptors (A1Rs) on the presynaptic axon terminals, decreasing GABA release. We demonstrated, in mice, that activation of cannabinoid-1 receptors (CB1R) with the agonist WIN 55,212-2 (WIN) reduced the miniature GABA receptor-mediated postsynaptic current (mGPSC) frequency by a mechanism that requires astrocytes and A1R. WIN activated an intracellular Ca2+ signaling pathway in astrocytes. Activating this intracellular Ca2+ pathway with designer receptors exclusively activated by designer drugs (DREADDs) also decreased the mGPSC frequency and required A1R activation. The frequency of spontaneous Ca2+ events, including those induced by depolarization of a postsynaptic SCN neuron, was reduced by blocking CB1R activation with AM251, demonstrating neuronal endocannabinoid signaling modulates astrocytic Ca2+ signaling in the SCN. Finally, daytime application of WIN or adenosine phase advanced the molecular circadian clock, indicating that this cannabinoid signaling pathway is vital for the timing of circadian rhythms.

Gonadotropin-releasing hormone (GnRH) neurons control anterior pituitary, and thereby gonadal, function. GnRH neurons are active before outward indicators of puberty appear. Prenatal androgen (PNA) exposure mimics reproductive dysfunction of the common fertility disorder polycystic ovary syndrome (PCOS) and reduces prepubertal GnRH neuron activity. Early neuron activity can play a critical role in establishing circuitry and adult function. We tested the hypothesis that changing prepubertal GnRH neuron activity programs adult GnRH neuron activity and reproduction independent of androgen exposure in female mice. Activating (3Dq) or inhibitory (4Di) designer receptors exclusively activated by designer drugs (DREADDs) were targeted to GnRH neurons using Cre-lox technology. In control studies, the DREADD ligand clozapine n-oxide (CNO) produced the expected changes in GnRH neuron activity in vitro and luteinizing hormone (LH) release in vivo CNO was administered to control or PNA mice between two and three weeks of age, when GnRH neuron firing rate is reduced in PNA mice. In controls, reducing prepubertal GnRH neuron activity with 4Di increased adult GnRH neuron firing rate and days in diestrus but did not change puberty onset or GABA transmission to these cells. In contrast, activating GnRH neurons had no effect on reproductive parameters or firing rate and did not rescue reproductive phenotypes in PNA mice. These studies support the hypothesis that prepubertal neuronal activity sculpts elements of the adult reproductive neuroendocrine axis and cyclicity but indicate that other PNA-induced programming actions are required for full reproductive phenotypes and/or that compensatory mechanisms overcome activity-mediated changes to mitigate reproductive changes in adults.

Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), are implicit in causing obesity. Mutations that reduce BDNF and TrkB expression are associated with obesity in humans and mice. Recently, it was reported that Bdnf gene deletion in the neurons of the paraventricular hypothalamus (PVH) caused positive energy balance and severe obesity in the form of hyperphagia, impaired adaptive thermogenesis, and decreased energy expenditure. Thus, we hypothesize that activation of these neurons will have the opposite effect and provide an opportunity for long-lasting obesity treatment. To specifically activate BDNF-expressing PVH (PVHBDNF) neurons, we injected Cre-dependent adeno-associated virus (AAV) expressing the excitatory DREADD hM3Dq bilaterally into the PVH of Bdnf2A-Cre/+ knock-in mice and then administered clozapine-N-oxide (CNO). Using this technique, we demonstrated that acute activation of these neurons rapidly decreased normal nocturnal feeding and fasting-induced feeding in male and female mice. At thermoneutral temperatures, acute activation also rapidly increased adaptive thermogenesis, increased core body temperature, increased locomotion, increased energy expenditure, and decreased respiratory exchange ratio (RER) in male and female mice. These observations indicate that acute stimulation of PVHBDNF neurons promotes negative energy balance and weight loss. However, the rapid decrease in RER after activation of PVHBDNF neurons was followed by a delayed and prolonged increase in RER that remained elevated for 3 d in female mice. Thus, although acute activation of PVHBDNF neurons promotes negative energy balance in the short term, long-term effects of activation include sexually dimorphic overcompensatory mechanisms that may promote positive energy balance in female mice.

The basolateral amygdala complex, which contains the lateral (LA) and basal (BA) subnuclei, is a critical substrate of associative learning related to reward and aversive stimuli. Auditory fear conditioning studies in rodents have shown that the excitation of LA pyramidal neurons, driven by the inhibition of local GABAergic interneurons, is critical to fear memory formation. Studies examining the role of the BA in auditory fear conditioning, however, have yielded divergent outcomes. Here, we used a neuron-specific chemogenetic approach to manipulate the excitability of mouse BA neurons during auditory fear conditioning. We found that chemogenetic inhibition of BA GABA neurons, but not BA pyramidal neurons, impaired fear learning. Further, either chemogenetic stimulation of BA GABA neurons or chemogenetic inhibition of BA pyramidal neurons was sufficient to generate the formation of an association between a behavior and a neutral auditory cue. This chemogenetic memory required presentation of a discrete cue, and was not attributable to an effect of BA pyramidal neuron inhibition on general freezing behavior, locomotor activity, or anxiety. Collectively, these data suggest that BA GABA neuron activation and the subsequent inhibition of BA pyramidal neurons play important role in fear learning. Moreover, the roles of inhibitory signaling differ between the LA and BA, with excitation of pyramidal neurons promoting memory formation in the former, and inhibition of pyramidal neurons playing this role in the latter.

Attraction to opposite-sex pheromones during rodent courtship involves a pathway that includes inputs to the medial amygdala (Me) from the main and accessory olfactory bulbs, and projections from the Me to nuclei in the medial hypothalamus that control reproduction. However, the consideration of circuitry that attributes hedonic properties to opposite-sex odors has been lacking. The medial olfactory tubercle (mOT) has been implicated in the reinforcing effects of natural stimuli and drugs of abuse. We performed a tract-tracing study wherein estrous female mice that had received injections of the retrograde tracer, cholera toxin B, into the mOT were exposed to volatile odors from soiled bedding. Both the anterior Me and ventral tegmental area sent direct projections to the mOT, of which a significant subset was selectively activated (expressed Fos protein) by testes-intact male (but not female) volatile odors from soiled bedding. Next, the inhibitory DREADD (designer receptors exclusively activated by designer drugs) receptor hM4Di was bilaterally expressed in the mOT of female mice. Urinary preferences were then assessed after intraperitoneal injection of either saline or clozapine-N-oxide (CNO), which binds to the hM4Di receptor to hyperpolarize infected neurons. After receiving CNO, estrous females lost their preference for male over female urinary odors, whereas the ability to discriminate these odors remained intact. Male odor preference returned after vehicle treatment in counterbalanced tests. There were no deficits in locomotor activity or preference for food odors when subject mice received CNO injections prior to testing. The mOT appears to be a critical segment in the pheromone-reward pathway of female mice.

The cerebellum communicates with brain areas critically involved in control of goal-directed behaviors including the prefrontal and orbitofrontal cortices and midbrain and basal ganglia structures. In particular, the posterior cerebellum is important for cognitive flexibility and has been implicated in alcohol and drug-related memory. We hypothesized that the cerebellum, through its multiple connections to reward-related brain circuitry, regulates alcohol consumption. To test this, we expressed inhibitory designer receptors exclusively activated by designer drugs (DREADDs) in molecular layer interneurons (MLIs) in anterior (IV-V) or posterior (VI-VIII) cerebellar lobules of male and female mice and activated them during alcohol drinking sessions. In a home-cage drinking paradigm, alcohol consumption was significantly decreased by clozapine-N-oxide (CNO) or deschloroclozapine (DCZ) administration in male mice expressing DREADDs in posterior but not anterior lobules. CNO/DCZ injections did not affect drinking in DREADD expressing female mice or in male mice expressing the control vector. Activation of DREADDs expressed in anterior or posterior lobules had no effect on sucrose or quinine consumption in male or female mice. During operant self-administration sessions, DCZ decreased the number of licks and bouts in male but not female mice expressing DREADDs in posterior lobules with no effect in control vector mice. Performance on an accelerated rotarod was unaffected by chemogenetic manipulation while distance traveled in the open field was decreased by DREADD activation in anterior but not posterior lobules. These results indicate that neuronal activity within the posterior cerebellar cortex plays an important role in the control of alcohol consumption in male mice.

G-protein-coupled receptors (GPCRs) coupled to Gi signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile) in shaping the emergence of adult anxiodepressive behaviors and sensorimotor gating. To address the role of Gi signaling in these developmental windows, we used a CaMKIIα-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD (designer receptor exclusively activated by designer drugs) in forebrain excitatory neurons and enhanced Gi signaling via chronic administration of the DREADD agonist, clozapine-N-oxide (CNO) in the postnatal window (postnatal days 2-14) or the juvenile window (postnatal days 28-40). We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CaMKIIα-positive neurons in the forebrain, and that the administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker c-Fos. hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD-mediated inhibition of CaMKIIα-positive forebrain excitatory neurons did not alter anxiety- or despair-like behaviors in adulthood and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi signaling in CaMKIIα-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxiodepressive behaviors in adulthood.

The midbrain periaqueductal gray (PAG), particularly its ventrolateral column (vlPAG), is part of a key descending pathway that modulates nociception, fear and anxiety behaviors in both humans and rodents. It has been previously demonstrated that inhibitory GABAergic neurons within the vlPAG have a major role in this nociceptive modulation. However, the PAG contains a diverse range of neuronal subtypes and the contribution of different subtypes of inhibitory neurons to nociceptive control has not been investigated. Here, we employed a chemogenetic strategy in mice that express Cre recombinase under the promotor for the glycine transporter 2 (GlyT2::cre) to modulate a novel group of glycinergic neurons within the vlPAG and then investigate their role in nociceptive control. We show that activation of GlyT2-PAG neurons enhances cold and noxious heat responses and increases locomotor activity (LMA) in both male and female mice. In contrast, inhibition of GlyT2-PAG neurons reduced nociceptive responses, while locomotor behaviors were unaffected. Our findings demonstrate that GlyT2+ neurons in the vlPAG modulate nociception and suggest that strategies targeting GlyT2-PAG neurons could be used to design novel analgesic therapies.

Operant (instrumental) conditioning is a laboratory analog for voluntary behavior and involves learning to make a response for a reinforcing outcome. The prelimbic cortex (PL), a region of the rodent medial prefrontal cortex, and the dorsomedial striatum (DMS), have been separately established as important in the acquisition of minimally-trained operant behavior. Despite dense anatomical connections between the two regions, experimenters have only recently linked projections from the PL to the posterior DMS (pDMS) in the acquisition of an operant response. Yet, it is still unknown if these projections mediate behavioral expression, and if more anterior regions of the DMS (aDMS), which receive dense projections from the PL, are also involved. Therefore, we utilized designer receptors exclusively activated by designer drugs (DREADDs) to test whether or not projections from the PL to the aDMS influence the expression of operant behavior. Rats underwent bilateral PL-targeted infusions of either a DREADD virus (AAV8-hSyn-hM4D(Gi)-mCherry) or a control virus (AAV8-hSyn-GFP). In addition, guide cannulae were implanted bilaterally in the aDMS. Rats were tested with both clozapine-N-oxide (CNO) (DREADD ligand) and vehicle infusions into the aDMS. Animals that had received the DREADD virus, but not the control virus, showed attenuated responding when they received CNO microinfusions into the aDMS, compared to vehicle infusions. Patch clamp electrophysiology verified the inhibitory effect of CNO on DREADDs-expressing PL neurons in acute brain slices. GFP-expressing control PL neurons were unaffected by CNO. The results add to the recent literature suggesting that connections between the PL and aDMS are important for the expression of minimally-trained operant responding.

The median preoptic nucleus (MnPO) is a putative integrative region that contributes to body fluid balance. Activation of the MnPO can influence thirst, but it is not clear how these responses are linked to body fluid homeostasis. We used designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of the MnPO in drinking behavior and vasopressin release in response to peripheral angiotensin II (ANG II) or 3% hypertonic saline (3% HTN) in adult male Sprague Dawley rats (250-300 g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After two weeks' recovery, a subset of rats was used for extracellular recordings to verify functional effects of ANG II or hyperosmotic challenges in MnPO slice preparations. Remaining rats were used in drinking behavior studies. Each rat was administered either 10 mg/kg of exogenous clozapine-N-oxide (CNO) to inhibit DREADD-expressing cells or vehicle intraperitoneal followed by a test treatment with either 2-mg/kg ANG II or 3% HTN (1 ml/100-g bw, s.c.), twice per week for two separate treatment weeks. CNO-induced inhibition during either test treatment significantly attenuated drinking responses compared to vehicle treatments and controls. Brain tissue processed for cFos immunohistochemistry showed decreased expression with CNO-induced inhibition during either test treatment in the MnPO and downstream nuclei compared to controls. CNO-mediated inhibition significantly attenuated treatment-induced increases in plasma vasopressin compared to controls. The results indicate inhibition of CaMKIIa-expressing MnPO neurons significantly reduces drinking and vasopressin release in response to ANG II or hyperosmotic challenge.

Designer receptors exclusively activated by designer drugs (DREADD)-based chemogenetic tools are extensively used to manipulate neuronal activity in a cell type-specific manner. Whole-cell patch-clamp recordings indicate membrane depolarization, coupled with increased neuronal firing rate, following administration of the DREADD ligand, clozapine-N-oxide (CNO) to activate the Gq-coupled DREADD, hM3Dq. Although hM3Dq has been used to enhance neuronal firing in order to manipulate diverse behaviors, often within 30 min to 1 h after CNO administration, the physiological effects on excitatory neurotransmission remain poorly understood. We investigated the influence of CNO-mediated hM3Dq DREADD activation on distinct aspects of hippocampal excitatory neurotransmission at the Schaffer collateral-CA1 synapse in hippocampal slices derived from mice expressing hM3Dq in Ca2+/calmodulin-dependent protein kinase α (CamKIIα)-positive excitatory neurons. Our results indicate a clear dose-dependent effect on field EPSP (fEPSP) slope, with no change noted at the lower dose of CNO (1 µM) and a significant, long-term decline in fEPSP slope observed at higher doses (5-20 µM). Further, we noted a robust θ burst stimulus (TBS) induced long-term potentiation (LTP) in the presence of the lower CNO (1 µM) dose, which was significantly attenuated at the higher CNO (20 µM) dose. Whole-cell patch-clamp recording revealed both complex dose-dependent regulation of excitability, and spontaneous and evoked activity of CA1 pyramidal neurons in response to hM3Dq activation across CNO concentrations. Our data indicate that CNO-mediated activation of the hM3Dq DREADD results in dose-dependent regulation of excitatory hippocampal neurotransmission and highlight the importance of careful interpretation of behavioral experiments involving chemogenetic manipulation.

Until recently, hypocretin (Hcrt) neurons were the only known wake-promoting neuronal population in the lateral hypothalamus (LH), but subpopulations of inhibitory neurons in this area and glutamatergic neurons in the nearby supramammillary nucleus (SuM) have recently been found that also promote wakefulness. We performed chemogenetic excitation of LH neurons in mice and observed increased wakefulness that lasted more than 4 h without unusual behavior or EEG anomalies. The increased wakefulness was similar in the presence or absence of the dual orexin receptor blocker almorexant (ALM). Analysis of hM3Dq transfection and c-FOS expression in LH inhibitory neurons and in the SuM failed to confirm that the increased wakefulness was due to these wake-promoting populations, although this possibility cannot be completely excluded. To evaluate the relationship to the Hcrt system, we repeated the study in Orexin-tTA mice in the presence or absence of dietary doxycycline (DOX), which enabled us to manipulate the percentage of Hcrt neurons that expressed hM3Dq. In DOX-fed mice, 18% of Hcrt neurons as well as many other LH neurons expressed hM3Dq; these mice showed a profound increase in wake after hM3Dq activation even in the presence of ALM. In mice switched to normal chow, 62% of Hcrt neurons expressed hM3Dq along with other LH cells; chemogenetic activation produced even more sustained arousal which could be reduced to previous levels by ALM treatment. Together, these results indicate an LH neuron population that promotes wakefulness through an Hcrt-independent pathway that can act synergistically with the Hcrt system to prolong arousal.